The determination of enzyme mechanisms is a central topic in the study of biomolecular systems because they are involved in most processes in living organisms. The development of new experimental and computational biophysics methods that allow new and ever more detailed views of these processes is of fundamental importance not just from the basic science point of view, but also due to the wide range of applications of the methods and the knowledge derived from them. The goal of the proposal is to create a molecular movie where the position, movement and energy of every atom in the system followed over the course of the reaction. This will be achieved by pursuing two Specific Aims: (i) Time-Resolved Laue Crystallography of HMG-CoA Reductase (HMGR) and (ii) Simulation of the Reaction Pathway of HMGR. The application of the methodology will allow access to a level of detailed knowledge about enzyme chemistry that was not attainable up to now. The proposed approach relies on the emerging convergence of the timescales accessible by time- resolved crystallography and computational methods. We will use our recently developed photocaged cofactors to generate structural snapshots with a time resolution of 1-100 s by Laue crystallography. The ensembles of intermediate states generated by these snapshots will be deconvoluted using singular value decomposition (SVD) and connected using long timescale molecular dynamics (MD) simulations to provide structural, dynamic, and energetic insights into the complete reaction pathway. Polarizable and non- polarizable transition state force fields (TSFF) will be generated by the quantum-guided molecular mechanics (Q2MM). The use of TSFFs is 102-104 times faster than the widely used QM/MM methods, thus allowing extensive sampling, and treats the entire system at a consistent level, thus preventing the well- known problems resulting from the QM/MM interface region. Iterative cycles of crystal structure -> MD simulation -> Markov State ensemble generation -> SVD analysis of Laue data -> new time resolved structures will be used to study a complex reaction pathway, which can be broken down into smaller steps to facilitate both experimental and computational approaches. This combined methodology will be applied to the case of Pseudomonas mevalonii HMGR, which has a complex reaction mechanism involving three chemical steps, six large-scale conformational changes and two cofactor exchange steps. HMGR is of broad biomedical interest because it is the target of the widely used statins and a potential target for antibacterial treatments by new classes of antibiotics, but the methodology developed in this proposal is in principle applicable to a wide range of systems. To promote the use of the experimental and computational innovations introduced, all tool compounds and computational codes to be developed will be made available to the broader scientific community.